Many different chemical, biochemical, and other reactions include thermal cycling. Examples of thermal processes in the area of genetic amplification include, but are not limited to, Polymerase Chain Reaction (PCR), Sanger sequencing, etc. The reactions may be enhanced or inhibited based on the temperatures of the materials involved. Although it may be possible to process samples individually and obtain accurate sample-to-sample results, individual processing can be time-consuming and expensive.
One approach to reducing the time and cost of thermally processing multiple samples is to use a device including multiple chambers in which different portions of one sample or different samples can be processed simultaneously. When multiple reactions are performed in different chambers, however, one significant problem can be accurate control of chamber-to-chamber temperature uniformity. The need for accurate temperature control may manifest itself as the need to maintain a desired temperature in each of the chambers, or it may involve a change in temperature, e.g., raising or lowering the temperature in each of the chambers to a desired setpoint. In reactions involving a change in temperature, the speed or rate at which the temperature changes in each of the chambers may also pose a problem. For example, slow temperature transitions may be problematic if unwanted side reactions occur at intermediate temperatures. Alternatively, temperature transitions that are too rapid may cause other problems. As a result, another problem that may be encountered is comparable chamber-to-chamber temperature transition rate.
Another problem that may be encountered in those reactions in which thermal cycling is required is overall speed of the entire process. For example, multiple transitions between upper and lower temperatures may be required. Alternatively, a variety of transitions (upward and/or downward) between three or more desired temperatures may be required. In some reactions, e.g., polymerase chain reaction (PCR), thermal cycling must be repeated up to thirty or more times. Typical thermal cycling devices and methods that attempt to address the problems of chamber-to-chamber temperature uniformity and comparable chamber-to-chamber temperature transition rates, however, typically suffer from a lack of overall speedxe2x80x94resulting in extended processing times that ultimately raise the cost of the procedures.
One or more of the above problems may be implicated in a variety of chemical, biochemical and other processes. Examples of some reactions that may require accurate chamber-to-chamber temperature control, comparable temperature transition rates, and/or rapid transitions between temperatures include, e.g., the manipulation of nucleic acid samples to assist in the deciphering of the genetic code. See, e.g., J. Sambrook and D. W. Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory (2001). Nucleic acid manipulation techniques include amplification methods such as polymerase chain reaction (PCR); target polynucleotide amplification methods, such as self-sustained sequence replication (3SR); methods based on amplification of probe DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR); transcription-based methods, such as ligation activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA); and various other amplification methods, such as repair chain reaction (RCR) and cycling probe reaction (CPR).
One common example of a reaction in which all of the problems discussed above may be implicated is PCR amplification. Traditional thermal cycling equipment for conducting PCR uses polymeric microcuvettes that are individually inserted into bores in a metal block. The sample temperatures are then cycled between low and high temperatures, e.g., 55xc2x0 C. and 95xc2x0 C. for PCR processes. When using the traditional equipment according to the traditional methods, the high thermal mass of the thermal cycling equipment (which typically includes the metal block and a heated cover block) and the relatively low thermal conductivity of the polymeric materials used for the microcuvettes result in processes that can require two, three, or more hours to complete for a typical PCR amplification.
Another problem experienced in the preparation of finished samples (e.g., isolated or purified samples of, e.g., nucleic acid materials such as DNA, RNA, etc.) of human, animal, plant, or bacterial origin from raw sample materials (e.g., blood, tissue, etc.) is the number of thermal processing steps and other methods that must be performed to obtain the desired end product (e.g., purified nucleic acid materials). In some cases, a number of different thermal processes must be performed, in addition to filtering and other process steps, to obtain the desired finished samples.
One example is in the preparation of a finished sample (e.g., purified nucleic acid materials) from a starting sample (e.g., a raw sample such as blood, bacterial lysate, etc.). To obtain a purified sample of the desired materials in high concentrations, the starting sample must be prepared for, e.g., PCR, after which the PCR process is performed to obtain a desired PCR product. The PCR product is then subject to further manipulation such as sequencing, ligation, electrophoretic analysis, etc.
One method of improving conventional thermal cycling processes involves the use of electromagnetic radiation and energy absorbing pigments and dyes to absorb the radiation and convert it to thermal energy. The use of electromagnetic radiation absorbers such as pigments and dyes can interfere with reactions that involve the use of an enzyme. The enzyme can be deactivated, thereby preventing the formation of the desired products, e.g., PCR amplification products. Thus, there is a need for methods that allow for the use of electromagnetic radiation and absorbers such as dyes without adverse affects on the formation of the desired reaction products.
The present invention provides various compositions and methods that involve the use of an enzyme and a dye, such as a near-infrared (near-IR or NIR) dye. Such compositions and methods are preferably used for processing sample mixtures that include biological materials. Preferred methods involve the use of thermal cycling of a sample material that includes a biological material and an enzyme through the application of electromagnetic energy. A dye is used to convert the electromagnetic energy into thermal energy and a surfactant is used to inhibit (i.e., reduce, prevent, and/or reverse) interaction between the enzyme and the dye. As used herein, inhibiting interaction between the enzyme and the dye involves reducing the interaction compared to the same system when the surfactant is not present. Preferably, inhibiting interaction between the enzyme and the dye involves preventing the interaction from occurring and/or substantially completely reversing such interaction.
The present invention provides a composition that includes a near-IR dye and greater than about 0.5 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, wherein the composition is stable in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, and more preferably at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C. Preferably, the composition also includes an enzyme, which is a polymerase or a ligase.
The present invention also provides a composition that includes a near-IR dye, at least about 1 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, a polymerase enzyme, and a triphosphate (e.g., a dNTP), wherein the composition is stable in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C. Preferably, the near-IR dye is a cyanine dye or a diimminium dye.
A preferred composition of the present invention includes a near-IR dye selected from the group of a diimminium dye, a cyanine dye, and a mixture thereof, at least about 1 wt-% of a nonionic surfactant, a polymerase enzyme, and a triphosphate, wherein the composition is stable in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C.
Another preferred composition of the present invention includes a near-IR dye selected from the group of a cyanine dye, a diimminium dye, and a mixture thereof; at least about 1 wt-% of a nonionic surfactant selected from the group of esters of fatty acids and polyhydric alcohols, fatty acid alkanolamides, ethoxylated fatty acids, ethoxylated aliphatic acids, ethoxylated fatty alcohols, ethoxylated aliphatic alcohols, ethoxylated sorbitol fatty acid esters, ethoxylated glycerides, ethoxylated block copolymers with EDTA, ethoxylated cyclic ether adducts, ethoxylated amide and imidazoline adducts, ethoxylated amine adducts, ethoxylated mercaptan adducts, ethoxylated condensates with alkyl phenols, ethoxylated nitrogen-based hydrophobes, ethoxylated polyoxypropylenes, polymeric silicones, fluorinated surfactants, polymerizable surfactants, and mixtures thereof; a polymerase enzyme; and a triphosphate; wherein the composition is stable in a thermal cycling process comprising cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C.
The present invention also provides a method of stabilizing a composition that includes a near-IR dye in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C. The method includes adding at least about 1 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, to the composition. Herein, weight percentages are based on the total weight of the composition. Preferably, the composition also includes an enzyme, preferably, a polymerase enzyme. Preferably, the surfactant is a nonionic surfactant.
The present invention also provides a method of conducting a thermal process. The method includes providing a sample mixture that includes a biological material, a near-IR dye, and greater than about 0.5 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof; and directly heating the sample mixture to a second temperature higher than the first temperature; wherein the near-IR dye is stable under a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C. Preferably, the method further includes cooling the sample mixture and directly reheating the sample mixture in a thermal cycling process.
Yet another method is one that involves denaturing hydrogen-bonded molecules. The method includes providing a sample mixture that includes hydrogen-bonded molecules, a near-IR dye, and greater than about 0.5 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, at a first temperature; and directly heating the sample mixture to a second temperature higher than the first temperature effective to denature the hydrogen-bonded molecules; wherein the near-IR dye is stable under a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50xc2x0 C. and about 95xc2x0 C.
As used in connection with the present invention, xe2x80x9cthermal processingxe2x80x9d (and variations thereof) means controlling (e.g., maintaining, raising, or lowering) the temperature of sample materials to obtain desired reactions. As one form of thermal processing, xe2x80x9cthermal cyclingxe2x80x9d (and variations thereof) means sequentially changing the temperature of sample materials between two or more temperature setpoints to obtain desired reactions. Thermal cycling may involve, e.g., cycling between lower and upper temperatures, cycling between lower, upper, and at least one intermediate temperature, etc.
As used herein, xe2x80x9cdirectlyxe2x80x9d heating a sample mixture means that the sample mixture is heated from within as opposed to heated upon transfer of thermal energy from an external source (e.g., heated container).
A composition containing a dye is xe2x80x9cstablexe2x80x9d in a thermal cycling process (due to the presence of a surfactant) if the dye displays no more than about a 20% decrease in absorbance relative to a control, i.e., the same composition not exposed to a thermal cycling process.
These and other features and advantages of the methods and compositions of the invention are described below with respect to illustrative and preferred embodiments of the invention.